The LID entered into a CRADA with MedImmune in 2006 to develop live attenuated vaccines against human respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluena viruses (HPIV) serotypes 1, 2, and 3. This CRADA remains in place. These vaccines are intended for intranasal administration to infants and young children. The first priority is to develop a bivalent vaccine against the predominant RSV subgroup, called subgroup A (RSV-A), and HPIV3, the two more prevalent and important viruses in this group. Secondary objectives are to develop live-attenuated vaccines against HMPV, HPIV1, and HPIV2. RSV RSV-A may provide sufficient cross-protection against RSV-B, which is the second subgroup of RSV, or it may be necessary to develop an RSV-B vaccine component. Vaccine virus candidates are generated from cDNA by reverse genetics, providing well-defined vaccines. Stepwise clinical evaluation begins in adults (who are seropositive for these common viruses), followed by seropositive older children (typically 12-59 months of age), followed by seronegative younger children and infants (typically 6 -59 months of age, depending on the virus). For RSV and HPIV3, viruses are evaluated further in young infants, typically 1-3 months of age. The following is a summary of ongoing studies. RSV-A vaccine: Under the CRADA, Medimmune is conducting a Phase 1/2a immunogenicity study in young infants with a recombinant rRSV (MEDI-559) that was developed by LID and contains a set of five independent attenuating elements. In a previous phase 1 study by LID, an earlier version of this virus (that differs by 27 silent mutations and appears to be phenotypically indistinguishable) was well-tolerated and immunogenic in 1-2 month old infants. The purpose of the present study is to get further data on safety and immunogenicity. LID has initiated a clinical study of a live attenuated RSV bearing deletion of the M2-2 coding sequence (delM2-2). This virus exhibits up-regulated viral gene expression that may increase its immunogenicity. In an accompanying report, we describe the development of a version of MEDI-559 that has been modified to increase its genetic and phenotypic stability. This virus, called cps2, is being manufactured as clinical trial material for phase I studies. Other candidates are in pre-clinical development and will be described next year. RSV-B vaccine: We developed a reverse genetics system for RSV-B that can be used to develop vaccine candidates based on results of ongoing studies with RSV-A. HPIV3 vaccine: One strategy towards an HPIV3 vaccine has been to use reverse genetics to construct chimeric viruses that contain genes from both HPIV3 and bovine PIV3 (BPIV3). The LID previously showed that BPIV3 is attenuated and well-tolerated in non-human primates and seronegative children, and that the determinants of this host range restriction are polygenic and appear to be stable. The strategy in making chimeras of HPIV3 and BPIV3 is to combine the host range restriction phenotype of BPIV3 with the F and HN neutralization and major protective antigens of HPIV3. Two chimeras have been under clinical evaluation. The first is rB/HPIV3, which consists of the BPIV3 backbone in which the F and HN protective antigen genes have been replaced by those of HPIV3. The second chimera is rHPIV3-Nb, which consists of the HPIV3 backbone in which the N gene has been replaced by that of BPIV3. These two viruses have been evaluated in adults, HPIV3-seropositive children, and HPIV3-seronegative children >6 months of age. Both viruses were highly restricted in adults and seropositive children but readily infected seronegative children. The mean peak titers of shed virus for rB/HPIV3 and rHPIV3-Nb were 102.8 and 103.7 PFU/ml, respectively. Although rB/HPIV3 was thus more restricted in replication, it induced significantly higher serum antibody titers specific to HPIV3. These data suggest that the rB/HPIV3 vaccine is more attenuated and yet more immunogenic, and thus may be the preferred vaccine for further clinical development. The basis for the difference in the two viruses is unknown. As one possibility, the viral interferon antagonists in rB/HPIV3 are of bovine origin and may be less effective in inhibiting the interferon response in human cells, which in turn may have an adjuvant effect. A second strategy towards an HPIV3 vaccine is based on an attenuated version of HPIV3 called rHPIV3cp45. This is a recombinant version of a biologically derived cold-passaged (cp) virus that previously had been shown by LID and collaborators to exhibit satisfactory infectivity, safety, immunogenicity, and lack of transmissibility in seronegative infants and children. LID re-derived this virus from cDNA to provide a known pedigree for safety reasons, and the attenuating mutations were identified as described in previous reports. The vaccine is being evaluated in a 2-dose, double-blind, placebo controlled trial in HPIV3-seronegative children of 6-36 months of age. The two doses are spaced at a 6-month interval. This is designed to evaluate the durability of immunity in this population and to identify appropriate dosing intervals. This is being evaluated in companion protocols through Seattle Children's Hospital and the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Group. A total of 40 children have been enrolled. HPIV1 vaccine: We previously used reverse genetics to generate a live attenuated HPIV1 vaccine candidate called rHPIV1-C(R84G/del170)HN(T553A)L(Y942A) that is attenuated by defined mutations including ones that have been engineered for genetic stability. This virus was immunogenic and protective in African green monkeys (AGMs). When given to 35 unscreened healthy adult volunteers, only 4 individuals were infected and shedding was minimal and with no illness, which is the desired safety profile for an attenuated virus. When evaluated in seropositive children 15 to 59 months of age (double-blind, 10 vaccine recipients and 5 placebo), none of the vaccinees were detectably infected, consistent with a highly attenuated nature. The vaccine presently is being evaluated in seronegative children 6 to 59 months of age (N = 30, with a vaccine to placebo ratio of 2:1). Results are incomplete but indicate that vaccine virus infectivity and shedding were low. Thus, this virus may be over-attenuated, which may be corrected by removing one attenuating mutation. HPIV2 vaccine: We previously used reverse genetics to generate a live attenuated intranasal HPIV2 vaccine called rHPIV2-V94(15C)/948L/1724 that, like the HPIV1 candidate, included stabilized mutations. This virus was immunogenic and protective in AGMs. When given to 15 seropositive adult volunteers, only 4 were infected and vaccine virus was shed in minimal amounts. The virus is presently being evaluated in seropositive children 15 to 59 months of age (N = 15, with a vaccine to placebo ratio of 2:1). HMPV vaccine: LID evaluated a recombinant version of wild-type HMPV for infectivity and pathogenicity in adults. The virus was infectious but caused minimal disease, as would be expected in this seropositive population. This showed that the recombinant backbone was suitable as the foundation for developing live-attenuated derivatives. LID initiated the first HMPV vaccine clinical study. This involves a live-attenuated HMPV vaccine virus (rHMPV-Pa) in which the HMPV P gene was replaced by that of avian MPV, thus conferring a host range attenuation phenotype. This virus was administered successively to healthy adults and seropositive children >12 months of age, with the result that there was no detectable HMPV infection or shedding, consistent with this being a highly attenuated virus. The vaccine is presently being evaluated in seronegative children >6 months of age.